31 In Vitro Antimicrobial Activity of Crude Extracts of Erythrina abyssinica and Capsicum annum in Poultry Diseases Control in the South Western Agro-Ecological Zone of Uganda Charles Lagu 1 and Kayanja I.B. Frederick 2 1 Department of Biology, Faculty of Science, Mbarara University of Science and Technology, 2 Department of Anatomy, Faculty of Medicine, Mbarara University of Science and Technology, Mbarara 1. Introduction Many small holder farmers in the south western agro-ecological zone (SWAEZ) of Uganda have for a very long time been using medicinal plants especially Erythrina abyssinica and Capsicum annum for the management of worms, Newcastle disease and other microbial infections respectively in local poultry (Nsubuga-Mutaka et al., 2005 and Lagu and Kayanja (2010). Commonly the root barks of Erythrina abyssinica are picked crushed and mixed with water and administered to the birds. The ripped fruits of Capsicum annum are picked crushed and mixed with solutions of ash and water (ITDG and IIRR) (1996); Lagu and Kayanja (2010). These medicinal combinations are mainly given to birds to treat them against worms and other microbial infections and worms (ITDG and IIRR, 1996). The farmers have been using these medicinal plant extracts for a very long time Katunguka- Rwakishaya et al., 2004; Olila et al., 2007; Ejobi et al., (2007). It is however, not clear if the Erythrina abyssinica and Capsicum annum have activity against the common microbes that affect the poultry. This study aims to investigate the anti-microbial activities and minimum inhibitory concentrations (MICs) of Erythrina abyssinica and Capsicum annum used by the farmers in the control and treatment of common poultry infections in the south western agro-ecological zone of Uganda. 2. Materials and methods 2.1 Identification and description of medicinal plants (for Erythrina abyssinica and Capsicum annum) 2.1.1 Erythrina abyssinica (Leguminosae) Erythrina abyssinica (Leguminosae)is a species of leguminous tree as seen in Figure 1. It is distributed in Congo Republic (ex Belgian), the Sudanese Republic, Ethiopia, Eritrea, www.intechopen.com
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31
In Vitro Antimicrobial Activity of Crude Extracts of Erythrina abyssinica and Capsicum annum in
Poultry Diseases Control in the South Western Agro-Ecological Zone of Uganda
Charles Lagu1 and Kayanja I.B. Frederick2 1Department of Biology, Faculty of Science,
Mbarara University of Science and Technology, 2Department of Anatomy, Faculty of Medicine, Mbarara University of Science and Technology,
Mbarara
1. Introduction
Many small holder farmers in the south western agro-ecological zone (SWAEZ) of Uganda
have for a very long time been using medicinal plants especially Erythrina abyssinica and
Capsicum annum for the management of worms, Newcastle disease and other microbial
infections respectively in local poultry (Nsubuga-Mutaka et al., 2005 and Lagu and Kayanja
(2010). Commonly the root barks of Erythrina abyssinica are picked crushed and mixed with
water and administered to the birds. The ripped fruits of Capsicum annum are picked
crushed and mixed with solutions of ash and water (ITDG and IIRR) (1996); Lagu and
Kayanja (2010). These medicinal combinations are mainly given to birds to treat them
against worms and other microbial infections and worms (ITDG and IIRR, 1996). The
farmers have been using these medicinal plant extracts for a very long time Katunguka-
Rwakishaya et al., 2004; Olila et al., 2007; Ejobi et al., (2007). It is however, not clear if the
Erythrina abyssinica and Capsicum annum have activity against the common microbes that
affect the poultry. This study aims to investigate the anti-microbial activities and minimum
inhibitory concentrations (MICs) of Erythrina abyssinica and Capsicum annum used by the
farmers in the control and treatment of common poultry infections in the south western
agro-ecological zone of Uganda.
2. Materials and methods
2.1 Identification and description of medicinal plants (for Erythrina abyssinica and Capsicum annum)
2.1.1 Erythrina abyssinica (Leguminosae)
Erythrina abyssinica (Leguminosae)is a species of leguminous tree as seen in Figure 1. It is
distributed in Congo Republic (ex Belgian), the Sudanese Republic, Ethiopia, Eritrea,
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Uganda, Kenya, Tanzania and Zimbabwe. In northern and western Ethiopia, it is found at
elevations between 1600 and 2100 m. Erythrina abyssinica (Luganda name: Muyirigiti or
Jjirikiti; Runyakole name: Ekiko), is a deciduous savannah legume. It grows in open
woodland and grassland. It has characteristic red overflowing flowers. It can be propagated
through seedlings, cuttings and truncheons. In the south western rangelands of Uganda, it is
sometimes planted along fences of paddocks to support barbed wires. It has various
traditional medicinal applications in livestock. It is also used in traditional human medicine
Figure 2.
Fig. 1. Erythrina abyssinica tree
Fig. 2. Dry root barks of Erythrinna abyssinica
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2.1.2 Capsicum annum (solanaceae)
Capsicum annum (solanaceae) was identified in the study areas by a botanist from Mbarara University of Science and Technology. Capsicum annum belongs to the kingdom plantae plants, subkingdom of tracheobiota (vascular plants), super-division of spermatophyta (seed plants), division of magnoliophyta (flowering plants), class magnoliopsida (dicotyledons), subclass, asteridae, order, solanels, family of solanaceae (potato family), genus capsicum L. (pepper), species Capsicum annum L. (cavenne pepper) and variety Capsicum annum L. var annum (Cayenne pepper).
Capsicum annum is a perennial shrub growing up to 2 m (6’) in height, with woody a trunk? Its leaves have various shapes usually elliptical up to 10 cm (4”) long as seen in Figure 1. The flowers are white to yellowish in groups of 2 or 3 followed by small, upright, fiery, green fruits that ripen to red. The active ingredient in the plant is capsaicin that is used for management of various medical conditions. The varieties of this “fruit” vary greatly in size, color and pungency. The plant extract that provides therapeutic action is the seed oil.
Fig. 3. Capsicum annum plant
2.2 Sample collection and post harvest handling (for Erythrina abyssinica and Capsicum annum)
Information gathered included vernacular names and parts used in the preparation of herbal remedies. The plants were identified by botanist from Mbarara University of Science and Technology. Voucher specimens were deposited in the University. Fresh samples of the plant materials (root barks, stem barks and leaves) of Erythrina abyssinica were collected from 500m away from Rubare town in Ntungamo district on 19th May, 2010. Four (4) km away from Rubindi sub county in Mbarara district on 22nd May, 2010. Seven (7) km away from Bugongi sub county in Bushenyi district on 23rd May, 2010 and 4 km away from Lwanda Sub County GPS location S00040.379’; E031028.7779, Rakai district on 16th June, 2010. The seeds and leaves of Capsicum annum were collected during early morning to late afternoon and placed in a plastic bag and stored in the vehicle
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During field collection of the samples, the sites where the plants were found were
geographically and ecologically described. Ejobi et al., 2007 found out that these plants were
abundantly found in all areas in the SWAEZ. The quantity of each plant biomass collected
depended on approximately with the amount of plant biomass needed to yield enough
concentrates for in vitro studies. The plant materials were kept in a plant press in the
department of Biology, Mbarara University of Science and Technology. The plants were
dried at room temperature at Mbarara Zonal Agricultural Research and Development
Institute (Mba ZARDI). The partially dried plants samples were collected and packed in a
plastic bag and then transported to Natural Chemothepeutics Research laboratories (NCRL)
Wandegeya, Kampala for extraction and concentration of plant extracts.
The concentrated samples were taken to the Microbiology and Parasitology Laboratory of
the School of Veterinary Medicine, Makerere University for the activity studies. Voucher
specimens of the plants collected were as follows; Root barks Ntu001Ea, Bus 002Ea, Mbra
003 Ea, Rakai 004 Ea; the stem barks included Ntu005Ea, Bus 006Ea, Mbra 007Ea, Rakai 008
Ea. The leaves sample include Ntu009Ea, Bus 010Ea, Mbra 011Ea, Rakai 012 Ea.
The Voucher specimens for Capsicum annum viz; Leaves Ntu001Ca, Bus002Ca, Mbra003Ca,
Rakai 004Ca. Fruits Ntu005Ca, Bus006Ca, Mbra007Ca, Rakai008Ca of the plants studied was
kept in the departmental laboratory.
Drying and milling
The leaves, root bark and stem bark of Erythrina abbysinica and the leaves and fruits of
Capsicum annum from Mbarara, Bushenyi, Ntungamo and Rakai districts were dried at 50-
60oC in a vacuum oven for 24 hours. The dry plant material samples were milled using an
electric grinder in fine particles.
2.3 Obtaining process of crude extracts of the plant parts used, including the determination of ‘extract yield” and “Standardization of dosages”
The leaves, root bark and stem bark of Erythrina abbysinica leaves and fruits of Capsicum
annum
2.3.1 Extraction, filtration and concentration
The milled samples (100-500g) were soaked in 70% ethanol (5L) for 48hours with frequent
shaking. Thereafter the extracts were filtered first with cotton wool followed by Whatman
filter paper® and stored in at room temperature. The filtrate was then concentrated using
vacuum rotary evaporator. The concentrates were then dried in vacuum oven at 60oC to
dryness.
2.3.2 Determination of extract yield
The percentage yield of the extract was determined gravimetrically using the dry weight of
extract (x) and soaked samples material (y) as follows
Percentage yield = x/y * 100 (1)
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2.3.3 Standardization of dosages
The information gained on the percent yields of crude extracts was used for standardizing
dosage rates of fine powder preparations of the plant materials. For example, the amount of
crude extract contained in a known weight of fine powder of plant materials was calculated
from the formulae given above.
2.4 Determination of antimicrobial activity of the leaves, root bark and stem bark of Erythrina abbysinica, the leaves and fruits of Capsicum annum
The concentrated extracts of the roots, stem and leaves of Erythrina abyssinica and Capsicum
annum, from the National Chemotherapeutics Laboratory, Wandegeya were transported to
the department of microbiology laboratory at the School of Veterinary Medicine, Makerere
flame; Micro pipettor (10µl-200µl), adjustable; Spreader/wire loop; Agar borer and
Incubator.
2.4.2 Method antimicrobial activity
Approximately 2-5 freshly grown bacterial colonies of the test organism were emulsified
into Nutrient broth and incubated for 10-15 minutes at room temperature. With a spreader
of wire loop, Mueller Hinton agar plate was evenly inoculated and plate allowed to stand
for 5 minutes at room temperature (i.e. 25oC).
Using a sterile agar borer (Sterilized using a bunsen flame), wells were dug into the
inoculated agar at reasonable distance apart (approximately 5 cm).
The plant extract(s) were transferred into the created agar wells till when full. Extract were
not allowed to float on the agar surface. The plate lid was replaced and did not turn the
petri-dish upside down. The setup was incubated at 37oC overnight. The presence for
bacterial inhibition zones around each well looked for. Appearance of clear zones around a
well was indicative of the anti bacterial activity of an extracts (Bizimenyera et al., 2005).
2.4.3 Determination of the Minimum Inhibitory Concentration (MIC) of extract
2.4.3.1 Materials for Minimum Inhibitory Concentration (MIC) of extract
Broth culture of test organism (s); known concentration of plant extracts (e.g. 0.5g/ml,
1g/ml etc); set of test tubes; micro-pipettor (adjustable 100µl-1000µl); Nutrient broth; Sterile
physiological saline; incubator.
2.4.3.2 Method for Minimum Inhibitory Concentration (MIC) of extract
A set of test tubes were dispensed 0.5 ml (500µl) of physiological saline. An equal volume
(i.e. 0.5ml) of test plant extract were added to the saline in first tube and mixed the two
thoroughly well.
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This was repeated throughout all the test tube and the last aliquot 0.5 ml of solution from
the last tube discarded so as to have uniform volume. This constituted a two fold serial
dilution whereby each step moved to the right reduced the concentration of the extract by a
factor of 2. About 100µl of a 24 hour culture of the test organism were added to each of the
test tubes (containing the already serially diluted extract). This was mixed thoroughly well,
plugged with cotton wool and incubated the preparation at 37oC for 16-24 hours.
At microbiology laboratory, the samples were each weighed and dissolved in
Dimethylesulfoxide (DMSO) at a final concentration of 0.5g/ml. Mueller Hinton Agar
(MHA) plates for antibiotic sensitivity testing were prepared and inoculated with pure
colonies of E. coli, Staphylococcus spp., Streptococcus spp., Salmonella spp., and Pseudomonas spp.,
which were known to be the common causes of poultry diseases.
The wells were bored in the inoculated plates and the samples from the extracts were
impregnated into the wells and incubated overnight. A control plate inoculated with E. coli
and impregnated with Dimethylesulfoxide (DMSO) and a known antibiotic, Ciprofloxacin
was set up as negative and positive controls respectively.
After 24hrs of incubation, the plates were examined for antibacterial activity on the different
sample extracts, and the results were as follows:
2.4.4 Observation/ interpretation of results
The inoculated test tubes were examined for inhibition of growth, where there is
antibacterial activity; there was inhibition of growth, thus no turbidity in the test tube. The
minimum inhibitory concentration (MIC) of a compound (extract) in this case the least
concentration to have inhibited bacterial growth.
2.5 Data analysis
Data collected were entered in Excel windows 2007 (Microsoft Corporation). Frequencies,
means and graphs were derived to explain phenomenon on significance and relationships to
activities and minimum inhibitory concentrations of crude extracts of root, stem barks and
leaves of Erythyrina abyssinica and leaves and leaves and fruits of Capsicum annum.
2.6 Results
2.6.1 Crude extracts of root, stem barks and leaves of Erythrina abyssinica
It is clear from Figure 4 and Table 1 that root barks and stem barks of Erythrina abbyssinica
have better yields than the leaves.
In-vitro microbiological studies on Erythrinna abbyssinica indicate that root and stem bark
extracts have activities against Staphyloccus aureus in Ntungamo, Mbarara, Bushenyi and
Rakai. Pseudomonas auroginosa in Mbarara, Bushenyi except leaves from Bushenyi. No
activities were noted on E. coli and Salmonella species as detailed in table 2 and Figure 5.
It has been demonstrated that root and stem barks have activity against Staphylococcus aureus
in all districts except stem bark for Rakai. The root barks, stem barks and leaves of Mbarara,
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Bushenyi respectively were effective against Pseudomonas aeruginosa demonstrated in Table 3.
The study found that the leaves extracts did not have antimicrobial activities.
Plant Sample Location Plant part Weight of sample (g)
Dry weight of concentrate (g)
% yield
E. abbysinica
Ntungamo Root barks 450.0 39.6 8.8
Mbarara Root barks 295.6 37.8 12.8
Bushenyi Root barks 689.5 53.0 7.7
Rakai Root barks 500.0 24.3 4.9
Ntungamo Stem barks 537.0 11.6 2.2
Mbarara Stem barks 435.8 10.7 2.5
Bushenyi Stem barks 483.7 13.4 2.8
Rakai Stem barks 500.0 44.6 8.9
Ntungamo Leaves 500.1 3.2 0.6
Mbarara Leaves 451.7 3.2 0.7
Bushenyi Leaves 761.6 29.4 3.9
Rakai Leaves 500.0 31.1 6.2
Table 1. Percentage yield extract of leaves, root bark and stem bark of Erythrina abbysinica from Mbarara, Bushenyi, Ntungamo and Rakai districts
Fig. 4. Percentage yield determination of plant extracts (Erythrinna abbyssinica)
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Antimicrobial assay of plant extracts against selected bacteria as indicated below
Inhibition zone (mm)
No. Identity/ Name of
Extracts E. coli Salmonella
Staphlococcus aureus
Pseudomonas aeruginosa
District
1 Root bark Ea Ntungamo 0 0 13 0 Ntungamo
2 Root bark Ea Bushenyi 0 0 14 0 Bushenyi
3 Root bark Ea Mbarara 0 0 12 8 Mbarara
4 Root bark Ea Rakai 0 0 15 0 Rakai
5 Stem bark Ea Ntungamo 0 0 11 0 Ntungamo
6 Stem bark Ea Bushenyi 0 0 12 7 Bushenyi
7 Stem bark Ea Mbarara 0 0 14 0 Mbarara
8 Stem bark Ea Rakai 0 0 0 0 Rakai
9 Leaves Ea Ntungamo 0 0 0 0 Ntungamo
10 Leaves Ea Bushenyi 0 0 0 8 Bushenyi
11 Leaves Ea Mbarara 0 0 0 0 Mbarara
12 Leaves Ea Rakai 0 0 0 0 Rakai
Table 2. Antimicrobial assay of Erythrina abyssinica plant extracts against selected bacteria
Fig. 5. Antimicrobial assay of plant extracts from four districts against selected media
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Minimum Inhibitory concentration of different
micro-organisms (g/ml)
No. Identity/ Name
of Extracts
Original
sample
concentration
(g/ml)
E. coli Salmonella
Staphylo-
coccus
aureus
Pseudo-
monas
aeruginosa
1 Root bark Ea
Ntungamo 0.3 0 0 0.0047 0
2 Root bark Ea
Bushenyi 1 0 0 0.0313 0
3 Root bark Ea
Mbarara 1 0 0 0.0313 1
4 Root bark Ea
Rakai 1 0 0 0.0156 0
5 Stem bark Ea
Ntungamo 0.25 0 0 0.0039 0
6 Stem bark
Ea Bushenyi 0.41 0 0 0.0256 0.41
7 Stem bark
Ea Mbarara 0.224 0 0 0.0035 0
8 Stem bark
Ea Rakai 1 0 0 0 0
9 Leaves
Ea Ntungamo 0.39 0 0 0 0
10 Leaves
Ea Bushenyi 1 0 0 0 1
11 Leaves
Ea Mbarara 0.412 0 0 0 0
12 Leaves Ea Rakai 1 0 0 0 0
Table 3. Minimum Inhibitory concentration of different micro-organisms
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2.6.2 Crude extracts of leaves and fruits of Capsicum annum
The yields of Capsicum annum leaves and fruits are detailed in Figure 4. It is clear from the
tabular representation that leaves had better yield than fruits.
Studies on Capsicum annum indicate that fruits extracts of Mbarara have activities on Salmonella species and Psudomonas auroginosa for the case of fruit extracts from Rakai. No microbial activities were noted against Staphylococcus aureus and E.coli. It was demonstrated that leaves extracts have no microbial activities as illustrated in Table 5 and Figure 7.
Fig. 6. Percentage yield determination for the plant Capsicum annum
Results indicated that leaves of Capsicum annum have better yields than fruits of Capsicum
annum in all the four districts Mbarara, Ntangamo, Rakai and Bushenyi.
Antimicrobial assay of plant extracts against selected bacteria as indicated below
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Inhibition Zone (mm)
No. Identity/ Name of
Extracts E. coli Salmonella
Staphylococcus aureus
Pseudomonas aeruginosa
District
1 Leaves Ca Ntungamo
0 0 0 0 Ntungamo
2 Leaves Ca Bushenyi 0 0 0 0 Bushenyi
3 Leaves Ca Mbarara 0 0 0 0 Mbarara
4 Leaves Ca Rakai 0 0 0 0 Rakai
5 Fruits Ca Ntungamo 0 0 0 0 Ntungamo
6 Fruits Ca Bushenyi 0 0 0 0 Bushenyi
7 Fruits Ca Mbarara 0 8 0 0 Mbarara
8 Fruits Ca Rakai 0 0 0 7 Rakai
Table 5. Antimicrobial assay of Capsicum annum plant extracts against selected bacteria
Fig. 7. Antimicrobial assay of plant extracts against selected bacteria.
It was demonstrated that root and stem barks had activity against Staphylococcus aureus and
Pseudomonas aeruginosa demonstrated in Table 3.
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Sample ID Original sample
concentration (g/ml)
MICs of Extracts to different microorganisms (g/ml)
Staphylococcus aureus
Pseudomonas aeruginosa
Salmonella E.coli
Leaves Ca Ntungamo
1 0 0 0 0
Leaves Ca Bushenyi
1 0 0 0 0
Leaves Ca Mbarara
1 0 0 0 0
Leaves Ca Rakai 1 0 0 0 0
Fruits Ca Ntungamo
0.328 0 0 0 0
Fruits Ca Bushenyi
1 0 0 1 0
Fruits Ca Mbarara
1 0 0 0 0
Fruits Ca Rakai 1 0 1 0 0
Table 6. Minimum inhibitory concentration of different micro-organism
Fruits of Capsicum annum exhibited minimum inhibitory concentration against Salmonella
species and Pseudomonas aeruginosa detailed in table 6.
2.7 Discussion
2.7.1 Crude extracts of root, stem barks and leaves of Erythrina abyssinica
Yields of the plants extracts
The results show that the yields of root barks perform better than the stem barks and leaf
extracts detailed in Table 1 and Figure 3. There is an average 8.6%, 5.5% and 2.9% yield for
the root barks, stem barks and leaves respectively in the districts of Ntungamo, Mbarara,
Bushenyi and Rakai. The information gained on the percent yields of crude extracts were
used for standardizing dosage rates of fine powder preparations of the plant materials. For
example, the amount of crude extract contained in a known weight of fine powder of plant
materials can then be calculated. This agrees with findings by (Ejobi F and Olila D 2004;
Olila et al., 2007).
Antibacterial activities of the plant extracts
The antimicrobial activity of crude extracts of root barks and stem barks of Erythrina
abyssinica had activity against Staphylococus aureus in Ntungamo, Bushenyi, Mbarara and
Rakai districts except stem bark from Rakai. There was activity against Pseudomonas
aeuroginosa in Mbarara and Bushenyi respectively as detailed in table 2 and figure 4.
Generally, leaves extracts showed no microbial activity except for leaf extracts from
Bushenyi district. No antibacterial activities were noted against Salmonella species and E.coli.
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The attributing factor for anti bacterial activity of the crude fruit extracts could be due to
presence of bioactive constituents’ in the extracts. This agrees with findings by Masola et al.,
2009 and Ogundare et al., (2006).
Moriyasu et al., (1998) collected stem bark E.abyssinica from Meru district of Kenya. They
carried structural elucidation of new flavanones isolated from E.abyssinica and found
presence of prenylated flavanones (abyssinin I(1), II (2), III(3), along with abyssinone V,
sigmoidin A, B, C and F, and sigmoidin B 4´-(methyl ether). These compounds exhibited
antimicrobial activities.
Further still, the presence of bioactive compounds viz; abyssinoflavanone IV, V and VI posses some antimicrobial activities as reported by (Ichimaru et al., 1996).
The stem woods of Erythrina latissima another species of Erythrina have two isoflavones
and a flavanone with isolates Isoflavone (erylatissin A, B, C) in addition to 10 known
flavonoids. These compounds exhibited antimicrobial activity against Escherichia coli,
Staphylococcus aureus, Bacillus subtilis and Candida mycoderma (Chacha et al., 2004). This
study found that Erythrina abyssinica was effective against Staphyloccus aureus and
Pseudomonas aeuroginosa.
The root and stem extracts of Erythrina abyssinica however did not show any microbial activity against Salmonella species and Escherichia coli.
A study by Masola et al.,(2009) in Mpwapwa, in the semi arid central zone of Tanzania
found out that bioactive constituents of terpenoids, tannins, phlobotannins, saponins and
cardiac glycosides were found to be present in the stem barks of the plant Adansonia digitata
(Bombacaceae) (African baobab). It would therefore mean similar bioactive compounds
were present in the plant Erythrina abysinnica.
Ikigai et al., (1993) indicated that purified tannins, saponins and terpenoids have anti
microbial activity. These bioactive compounds were reported to be effective against gram
positive and gram negative bacteria. They exert bactericidal and bacteriostatic effects. This
explained why there were activities on bacteria by the Erythrina abyssinica plant extracts.
Understanding the spectrum of antibacterial activity indicated that bioactive substances had
broad spectrum or narrow spectrum of activity. The degree of susceptibility (diameter of
inhibition zone) showed that gram positive bacteria were more susceptible compared to
gram negative bacteria. There was urgent need to undertake phytochemical analysis of
Erythrina abyssinica extracts to determine the chemical bioactive substances present in the
extracts.
Minimum inhibitory concentration (MICs)
The root and stem barks of Erythrina abyssinica had activity against Staphylococcus aureus in all districts except stem bark for Rakai. The root barks, stem barks and leaves of Mbarara, Bushenyi respectively were effective against Pseudomonas aeruginosa demonstrated in Table 3. This demonstrated that the leaves extracts do not have antimicrobial activities.
Traditionally, the results from in vitro antimicrobial tests are written as qualitative or quantitative. Qualitative results are reported as susceptible, intermediate or resistant,
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whereas quantitative results are reported as minimum inhibitory concentration (MIC) in µg/ml or g/ml or mg/l (Walker, 2006).
Walker,(2006), indicated that in-vitro antimicrobial susceptibility tests were predictive of in vivo therapeutic efficacy. However the ability of an in-vitro test to predict the clinical effectiveness of an antimicrobial agent is dependent on that test being performed correctly.
In vitro tests involve the continuous exposure of a relatively small concentration of bacteria to a constant level of antimicrobial agent under standardized testing conditions (Walker, 2006).
The selection of an appropriate dose can be driven by the result of quantitative susceptibility
tests (Ambrose, 2005). Studies in human medicine have demonstrated the clinical value of in
vitro susceptibility tests.
The interpretation of susceptibility testing depended on the relationship between in vitro
susceptibility and factors involved in relation to tissue drug concentration (which depended
on factors such as dose and pharmacokinetic and pharmaco-dynamic properties of the drug
or drug class.
2.7.2 Crude extracts of leaves and fruits of Capsicum annum
2.7.2.1 Yields of the plants extracts
This study found out that the yields of the leaves extracts of Capsicum annum were better
than the fruits as seen in Table 1 and Figure 2. There is an average 58% yield for the leaves
compared to 10-11% yields for the fruits in all the districts of Ntungamo, Mbarara,
Bushenyi and Rakai. The information gained on the percent yields of crude extracts are
used for standardizing dosage rates of fine powder preparations of the plant materials.
For example, the amount of crude extract contained in a known weight of fine powder of
plant materials can then be calculated. This agrees with findings by (Ejobi F and Olila D
2004; Olila et al., 2007).
2.7.2.2 Antibacterial activities of the plant extracts
The antimicrobial activity of crude extracts of fruits of Capsicum annum was positive for
Salmonella species and Pseudomonas aeuroginosa in Mbarara and Rakai respectively as detailed
in table 2. There were no microbial activity by leaves of Capsicum annum in all the districts of
Ntungamo, Mbarara, Bushenyi and Rakai. The fruit extracts did not have any activity on
Staphylococcus aureus and E.coli.
The attributing factor for anti bacterial activity of the crude fruit extracts could be due to
presence of bioactive constituents’ in the extracts. This agrees with findings by Masola et al.,
2009 and (Ogundare et al., 2006).
A study by Masola et al.,2009 in Mpwapwa, in the semi arid central zone of Tanzania found
out that bioactive constituents of terpenoids, tannins, phlobotannins, saponins and cardiac
glycosides were found to be present in the stem barks of the plant Adansonia digitata
(Bombacaceae) (African baobab).
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Further still, studies by Ikigai et al., 1993 indicate that purified tannins, saponins and
terpenoids have anti microbial activity. These bioactive compounds were reported to be
effective against gram positive and gram negative bacteria. They exert bactericidal and
bacteriostatic effects.
Understanding the spectrum of antibacterial activity will indicate if given bioactive
substance had broad spectrum or narrow spectrum of activity. The degree of susceptibility
(diameter of inhibition zone) will show that gram positive bacteria are more susceptible
compared to gram negative bacteria. The need to undertake phytochemical analysis of fruit
extracts of Capsicum annum to know the chemical bioactive substances present in these
extracts.
2.7.2.3 Minimum inhibitory concentration (MICs)
The fruit extracts of Capsicum annum from Bushenyi and Rakai exhibited minimum
inhibitory concentration against Salmonella species and Pseudomonas aeruginosa respectively as
detailed in table 4. There was no minimum inhibitory concentration noted for the case of
leaves of Capsicum annum.
Traditionally, qualitative and quantitative results from in vitro antimicrobial tests are
reported. Qualitative results are reported as susceptible, intermediate or resistant, whereas
quantitative results are reported as minimum inhibitory concentration (MIC) in µg/ml or
g/ml or mg/l (Walker, 2006).
Walker,(2006), noted that in-vitro antimicrobial susceptibility tests are predictive of in
vivo therapeutic efficacy. However the ability of an in-vitro test to predict the clinical
effectiveness of an antimicrobial agent is dependent on that test being performed
correctly.
Plant Sample
Location Plant part Weight of sample (g)
Dry weight of concentrate (g)
% yield
C. annum Mbarara Leaves 146.8 37.1 25.3
Bushenyi Leaves 62.4 53.9 86.4
Ntungamo Leaves 162.5 84.2 51.8
Rakai Leaves 253.0 174.0 68.8
Mbarara Fruits 85.3 8.8 10.3
Bushenyi Fruits 127.1 11.6 9.1
Ntungamo Fruits 61.3 4.9 8.0
Rakai Fruits 248.9 37.0 14.9
Table 4. Percentage yield extracts of the leaves and fruits of Capsicum annum from Mbarara, Bushenyi, Ntungamo and Rakai districts
In vitro tests cannot always predict the efficacy of an antibacterial agent in vivo (Walker, 2006). In vitro tests involve the continuous exposure of a relatively small concentration of bacteria to a constant level of antimicrobial agent under standardized testing conditions. The selection of an appropriate dose can be driven by the result of quantitative susceptibility tests (Ambrose, 2005). Despite these considerable differences, studies in human medicine have demonstrated the clinical value of in vitro susceptibility tests (Ambrose, 2005).
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A Bird's-Eye View of Veterinary Medicine
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By generating full range MICs, a laboratory can give clinicians information that may allow
them to individualize the therapeutic regimen, especially with regard to dose and dosing
frequency. For example, if the MIC is low, the dose or frequency of dosing may be
decreased. On the other hand, if the MIC is higher, but the organism is still considered
susceptible and the drug has a wide pharmacotoxicity margin, a higher dose of the drug
may be used.
Interpretation of susceptibility testing depends on knowing the relationship between in
vitro susceptibility and factors involved in relation to tissue drug concentration (which
depend on factors such as dose and pharmacokinetic and pharmaco-dynamic properties of
the drug or drug class.
3. Conclusions
The root and stem barks of E. abbyssinica have better yields than their leaves. The root and
stem barks exhibit antimicrobial activities against Staphylococus aureus and Pseudomonas
aeruginosa. The study had demonstrated that the leaves extracts generally did not have
antimicrobial activities. The MICs of the root and stem bark extracts ranged from (3.5-31.3)
mg/ml for Staphylococcus aureus and (410-1000) mg/ml for Pseudomonas aeruginosa. The
study agreed that farmers were right in using root barks of E. abyssinica to treat various
ailments in poultry diseases including other livestock diseases. There is need for further
research to do phyto-chemical analysis to analyze the bioactive constituents of the extracts,
undertake acute toxicity tests of the extracts.
It was clear that Capsicum annum have antibacterial activities against Salmonella species and
Pseudomonas aeruginosa. The results reveal that the fruit extracts of Capsicum annum have
activity against Salmonella species, Pseudomonas aeruginosa in Mbarara and Rakai districts
respectively. No activity was noted against E. coli and Staphylococcus aureus. The leaves of
Capsicum annum did not show any antibacterial activities. The MICs of the fruit extracts was
1000mg/ml. The study agreed that farmers may be right in using fruits of Capsicum annum
to treat various ailments in poultry diseases. There is need for further research to do
phytochemical analysis to analyze the bioactive constituents of the extracts, undertake acute
and chronic toxicity tests for Capsicum annum extracts.
4. Acknowledgements
We acknowledge the support Belgian Technical Cooperation (BTC) for funding this
research. We thank NARO for their assistance in transportation by availing means of
transport during plant collection process. Francis Umujal, Moses Agwaya and Henry
Tumusime staff of Natural Chemotherapeutics Research Laboratories (NCRL) Wandegeya
thanked for facilitating the process of extraction and concentration of plant extracts. We
applaud Mr. Nathan L. Musisi a staff of the Department of Microbiology and Parasitology,
Makerere University, Ms. Betty Ayoo Laura and Dr. Nsubuga Mutaka of Mbarara Zonal
Agriculture Research and Development Institute (Mba ZARDI) for assisting to get the work
done. We thank NARO for their assistance in transportation by availing means of transport
during plant collection process.
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In Vitro Antimicrobial Activity of Crude Extracts of Erythrina abyssinica and Capsicum annum in Poultry Diseases Control in the South Western Agro-Ecological Zone of Uganda
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5. References
Ambrose P (2005). Antimicrobial susceptibility breakpoints.PKPD and susceptibility break
points. Treat Respiratory Medicine (Suppl 4) 1:5
Bizimenyera S.E.,Swan G.E., Chikoto H&Eloff J N (2005). Rationale for using Peltophorum
africanum (Fabaceae) extracts in Veterinary Medicine. Journal of South African
Veterinary Association, 76:54-58.
Chacha M., Bojase-Moleta G and Majinda R.T.R (2004). Antimicrobial and radical
scavenging flavonoids from the stem wood of Erythrina latissima. Phytochemistry Vol
66, Issue 1, 2005, pp 99-104.
Ejobi F and Olila D (2004). On-farm validation of selected ethno-veterinary medical practices
in the Teso farming system. A technical report of NARO/DFID COARD. National
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How to referenceIn order to correctly reference this scholarly work, feel free to copy and paste the following:
Charles Lagu and Kayanja I.B. Frederick (2012). In Vitro Antimicrobial Activity of Crude Extracts of Erythrinaabyssinica and Capsicum annum in Poultry Diseases Control in the South Western Agro-Ecological Zone ofUganda, A Bird's-Eye View of Veterinary Medicine, Dr. Carlos C. Perez-Marin (Ed.), ISBN: 978-953-51-0031-7,InTech, Available from: http://www.intechopen.com/books/a-bird-s-eye-view-of-veterinary-medicine/in-vitro-antimicrobial-activity-of-crude-extracts-of-erythrina-abyssinica-and-capsicum-annum-in-poul